PROJECT SUMMARY: Human pluripotent stem cells (hPSCs) have the potential to differentiate into any cell type of the body. Deriving insulin-producing pancreatic beta cells from hPSCs holds great potential as a diabetes treatment. While recent advances have been made in generating stem cell-derived beta cells (SCBCs), these cells do not fully recapitulate mature beta cells in vitro, but do become more functionally mature after transplantation. This suggests that one or more components of the in vivo milieu, such as vascularization, hormone and growth factor signals, and/or extracellular matrix (ECM) interactions, aid in beta cell maturation. We seek to recapitulate an in vivo-like niche for terminal differentiation and maturation in vitro with the goal of improving the generation of functional SCBCs. The ECM is a network of proteins and polysaccharides, of a unique composition in each tissue, capable of guiding cell migration, morphology, function and differentiation. In adult human islets, ECM has been found to play a key role in cell survival and insulin secretion, while in fetal islets, ECM has also been associated with beta cell proliferation, migration, and maturation. These data suggest that ECM is important for beta cell and islet health. During islet isolation from the pancreas, much of the native ECM is destroyed, and the health and function of these islets is extremely limited in vitro. Moreover, in vitro-derived SCBCs possess scant amounts of ECM. An unsuitable microenvironment, due to impaired or deficient ECM in vitro, may be a barrier to successful islet and SCBC culture. Our lab is among the first to apply decellularization techniques to the human pancreas and isolate high quality ECM (hP-ECM). The isolation of hP-ECM and the development of state-of-the art quantitative mass spectrometry (MS) techniques introduces the ability to more comprehensively study the human pancreatic matrisome, which ultimately will provide insight to create mimetic constructs. As ECM has been implicated to play an important role in fetal and adult islets, and our preliminary data show differences in the abundance of some matrix proteins between fetal and adult samples, I propose in Aim 1 to analyze decelled ECM samples alongside native tissue, from four distinct age groups: fetal, juvenile (1-18 yrs), young adult (21-29 yrs), and older adult (55+ yrs) using MS with DiLeu mass tag labeling. To target islet ECM specifically, knowing the isolation process damages islet ECM, I will use modern MS imaging techniques in Aim 2 to quantitatively and comprehensively measure in situ human islet-specific ECM composition for the first time. Using pepsin-digested hP-ECM, our lab is the first to produce a spontaneously gelling hydrogel (hP-HG), which I have shown can be used in 3-D culture platforms as a natural scaffold for testing the effect of pancreatic ECM on differentiating SCBCs. In Aim 3, I will test whether hP-HG will improve the maturation and function of SCBCs during differentiation in culture. We expect this new knowledge to translate into useful applications in islet and stem cell culture and transplantation.